Establishment Of Mouse SPATA2 Gene Knockout Model And Study Of Its Phenotype On Testis And Related Mechanism

Sertoli cell is a kind of somatic cell in testis seminiferous tubules. Its main functions include providing a reliable environment for spermatogenesis and nourishing germ cells to proliferate and differentiate. Sertoli cells combined with each other tightly to form blood-testis barrier,and secreted kinds of cytokines which have important effects on spermatogenesis. There were investigations indicating that spermatogenesis-associated protein 2 (SPATA2) is high-expressed in Sertoli cells of rats and humans. But the protein's function in male reproduction system is still unclear. The present study indicated SPATA2 is high-expressed in mouse Sertoli cells. And SPATA2 knockout mice were generated through CRISPR/Cas9 gene editing technology for further studies about the differences between knockout and wildtype mice in male fertility. The relevant mechanism would also be analyzed. The main findings in the investigation include the following points:1. Real-time PCR and Western blot assays showed that SPATA2 is higher-expressed in adult mouse testis than in other tissues or in puberty testis. The results of immunohistochemistry indicated SPATA2 was localized at the cytoplasm of Sertoli cells in seminiferous tubules.2. The Spata2 gene mutant mice were generated through CRISPR/Cas9n gene-editing system,which is an improved method of CRISPR/Cas9. After analyzing the genomic information of mutant mice, we chose one of them which is most appropriate for further investigation to build a mutant line.Then homozygous individuals were generated after breeding two generations. PT-PCR, western blot and immunofluorescence were used to identify the efficiency of knockout. The results proved that the homozygous SPATA2 knockout (KO) mice were able to use for study of the gene function.3. The analysis of male SPATA2 knockout mice fertility indicated that their testis were smaller and lighter 1/3 relative to wildtype controls from littermates, and 4 month-old KO mice presented lighter seminiferous tubules and thinner seminiferous epithelium. Further study showed that the number of sperm in KO mice epididymis decreased 40 percent. And some abnormalities in sperm morphology and a decreasing in sperm activity were displayed in SPATA2 KO mice.4. Breeding assay showed the litter size of SPATA2 KO male mice decreased 2.36 in average relative to wildtype controls from littermates and the difference was significant. The fertilization assay in vivo also demonstrate the KO male mice presented an obviously weakened fertilization ability of sperm especially for mating with the superovulation females.5. Immunofluorescence indicated the number of PCNA positive cells in KO testis reduced significantly while the apoptosis didn't increase. Further test revealed that the spermatogonia marked by PLZF also reduced but without a significance, and the spermatocyte marked by SCP3 and ?H2A.X both had significant decreasing. These results gave a preliminary explanation about why SPATA2 KO mice showed a decreasing in the number of sperm.6. Considering SPATA2 is expressed in Sertoli cell, the number of Sertoli cell was identified both in developmental testis and in adult testis through counting the cells marked by Wtl in seminiferous tubules. And the conclusion is no variation being observed in Sertoli cell numbers when deleting SPATA2 in male mice. Then the biotin tracer assay was used to identify the integrity of blood-testis barrier. It has been found that the blood-testis barrier in SPATA2 KO mice was impaired. This results further revealed the reason of weakened proliferation and reduced number of germ cells in KO testis.7. Several Sertoli cell expressed genes were detected on mRNA level and inhibin alpha subunit was found increased in KO testis, which was revealed possibly to be the fundamental reason of the phenotypes. Meanwhile, it has been detected that the expression of FSH beta subunit in pituitary experienced a decreasing.In conclusion, this study indicated that SPATA2 expressed in the cytoplasm of Sertoli cells in mouse.And the SPATA2 global knockout mice were generated by using CRISPR gene-editing technology, And it has illuminated the deletion of SPATA2 would lead to an increasing expression of inhibin,which is a negative regulator of FSH, and a decreased expression of FSH?, so that the male reproduction system would be impaired and a reduced number of sperm was presented as well as an attenuated fertility.